Project 2: Project Summary Our goal is to understand how a burst- forming unit erythroid (BFU-E) progenitor integrates multiple environmental (e.g. low oxygen) and hormonal (e.g. cortisol, PPAR? agonists) signals and makes the decision to undergo a self-renewal or differentiative division; in particular we will elucidate the network of transcription factors and the genes regulated by these factors that control this key developmental decision. In Aim 1 we will characterize in detail the transcriptomes as BFU-Es initiate and undergo in culture self-renewal divisions in the presence of dexamethasone together or not with Prolyl Hydroxylase 2 (PH2) inhibitors or PPAR? agonists. Parallel ChIP-Seq analysis of BFU-E cells initiating self-renewal divisions will identify genes potentially directly regulated by the glucocorticoid receptor (GR), HIF1?, and PPAR?. We will use this information in multiple bioinformatic approaches to identify additional transcription factors, chromatin- modifying enzymes, other transcriptional regulatory proteins, and RNA-binding proteins that potentially regulate self-renewal. In this analysis we also anticipate identifying novel cell surface proteins for isolation of early, mid, and late BFU-Es to higher levels of purity. In Aim 2 we will use cultures of purified early BFU-Es and a combination of dye staining to count cell divisions and single cell RNA sequencing to determine whether BFU-E self-renewal divisions, as well as differentiation divisions toward the CFU-E, progress through successive discrete transcriptional states or form a continuum of states. These studies will define how transcriptional states and numbers of cell divisions are coordinated during BFU-E self-renewal divisions and also during differentiation toward CFU-Es. In mouse BFU-Es we showed that PPAR? co-occupies many chromatin sites with the glucocorticoid receptor (GR); these presumed distal enhancer regions are enriched for DNA binding motifs for Smad 2 and 9 other transcription factors, many of which are known to play important roles in self-renewal of different stem cells. In Aim 3 we will use gain- and loss- of-function approaches in vitro and in vivo to test whether Smad 2 and ~5 other selected novel gene regulatory proteins identified in Aims 1 and 2, are important for BFU-E self-renewal. By determining the genes regulated by these factors, as well as by mapping the binding sites of these transcription factors and RNA binding proteins that are functionally important in the self-renewal process, we can construct a wiring diagram of the important genes and proteins in the self-renewal network. In addition, our understanding of how self-renewal occurs in this population of transit amplifying cells will likely have a wide impact on understanding of the self-renewal process in other cell lineages. Finally, our understanding of the self renewal of BFU-E cells and the consequent increase in numbers of red cells produced from each BFU-E progenitor, will likely pave the way for new types of treatments of many Epo-resistant anemias, including Diamond-Blackfan anemia.